2.1. PLANT ELONGATION AND GROWTH
The plant cell elongation mechanism is a fundamental process with primary importance in plant-tissue development. Cell elongation requires relaxation of the rigid primary cell wall (Carpita and Gibeaut, 1993, Plant J. 3:1-30; Cosgrove, 1993, Plant Physiol. 102:1-6; Fry, 1988, The Growing Plant Cell Wall Chemical and Metabolic Analysis, Lonoman Scientific & Technical, New York; Roberts, 1994, Curr. Opin. Cell Biol. 6:688-694). Several mechanisms for this relaxation have been suggested, including the activities of endo-xyloglucan transferase (Nishitani and Tominaga, 1992, J. Biol. Chem. 267:21058-21064), xyloglucan endotransglycosylase (Fry et al., 1992, Biochem. J. 282:821-828) and expansins (McQueen-Mason and Cosgrove, 1995, Plant Physiol. 107:87-100). Endo-1,4-.beta.-glucanase (hereinafter, EGase) has been suggested to play an important role in the elongation process (Shoseyov and Dekel-Reichenbach, 1992, Acta Hort. 329:225-227; Verma et al., 1975, J. Biol. Chem.250:1019-1026).
Substantial evidence for the involvement of a 1,3-1,4-.beta.-glucan-specific enzyme in cell elongation was found in monocotyledons (Hatfield and Nevins, 1987, Plant Physiol. 83:203-207; Hoson and Nevins, 1989, Plant Physiol. 90:1353-1358 1989; Inouhe and Nevins, 1991, Plant Physiol. 96:426-431). EGase has been implicated in xyloglucan degradation during vegetative growth and fruit ripening (Hayashi, 1989, Ann. Rev. Plant Physiol. 40:139-168; Hayashi et al., 1984, Plant Physiol. 25:605-610). The activity of this enzyme could affect the generation of oligosaccharins, signaling molecules that are involved, among other things, in plant development and cell elongation (see for review, Darvill et al., 1992, Glycobiology 2:181-198).
To date, most of the EGase genes isolated have been studied in relation to fruit ripening (Cass et al., 1990, Mol. Gen. Genet. 223:76-86; Fischer and Bennett, 1991, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:675-703; Lashbrook et al., 1994, Plant Cell 6:1485-1493; Tucker et al., 1987, Plant Mol. Biol. 9:197-203) and abscission zones (Kemmerer and Tucker, 1994, Plant Physiol. 104:557-562; Tucker and Milligan, 1991, Plant Physiol. 95:928-933; Tucker et al., 1988, Plant Physiol. 88:1257-1262).
More recently, Wu et al. (1996, Plant Physiol. 110:163-170) cloned the EGase gene from pea and showed its expression to be induced by auxin in elongating epicotyls.
Endogenous regulation of cell elongation appears to be dominated by cell wall mechanics. This process is a result of the interaction between internal turgor pressure and the mechanical strength of the cell wall (reviewed by Steer and Steer, 1989, New Phytol. 111:323-358). Unlike most plant cells, the growth of pollen tubes and root hairs is restricted to the tip zone (reviewed by Cresti and Tiezzi, 1992, "Pollen tube emission organization and tip growth," in Sexual Plant Reproduction, pp. 89-97, eds. Cresti and Tiezzi, Springer-Verlag, Berlin). The growing region of pollen tubes consists of two distinct layers when fully mature. The inner layer consists mostly of callose-related molecules and the outer layer contains pectin, xyloglucan (XG), cellulose (at low levels and poor crystallinity) and other polysaccharides (reviewed by Steer and Steer, 1989, New Phytol. 111:323-358).
Xyloglucans (XGs) are linear chains of .beta.-(1-4)-D-glucan, but unlike cellulose, they possess numerous xylosyl units added at regular sites to the 0-6 position of the glucosyl units of the chain (reviewed by Carpita and Gibeaut, 1993, Plant J. 3:1-30). XG can be extracted by alkaline treatment and then bound again in vitro to cellulose (Hayashi et al., 1994, Plant Cell Physiol. 35:1199-1205).
XG is bound to cellulose microfibrils in the cell walls of all dicotyledons and some monocotyledons (reviewed by Roberts, 1994, Curr. Opin. Cell Biol. 6:688-694). The XG bound to the cellulose microfibrils cross-links the cell-wall framework.
Plant-cell expansion, including elongation, requires the integration of local wall-loosening and the controlled deposition of new wall materials. Fry et al. (1992, Biochem J. 282:821-828) and Nishitani and Tominaga (1992, J. Biol. Chem 267:21058-21064) purified xyloglucan endo-transglycosylase (XET) and endo-xyloglucan transferase (EXT), respectively. These two enzymes were shown to be responsible for the transfer of intermicrofibrillar XG from one segment to another XG molecule and thus, suggested to be wall loosening-enzymes.
However, McQueen-Mason et al. (1993, Planta 190:327-331) showed that XET activity did not correlate with in vitro cell all extension in cucumber hypocotyls.
The effect of XG on growing tissues has been extensively investigated. XG oligosaccharides, produced by partial digestion with .beta.-(1-4)-D-glucanase and referred to as "oligosaccharins", alter plant-cell growth (reviewed by ldington and Fry, 1993, Advances in Botanical Research 19:1-101). One such oligosaccharin, XXFG (XG9), antagonizes the growth promotion induced in pea stem segments by the auxin 2,4-D at a concentration of about 1 nM (York et al., 1984, Plant Physiol. 75:295-297; McDougall and Fry, 1988, Planta 175:412-416). On the other hand, at high concentrations (e.g,., 100 .mu.M) oligosaccharins promote the elongation of etiolated pea stem segments (McDougall and Fry, 1990, Plant Physiol. 93:1042-1048). The mode of action of oligosaccharins is still unknown.
Another type of cell wall-loosening protein, termed "expansin", was isolated by McQueen-Mason et al. (1992, The Plant Cell 4:1425-1433). Expansin does not exhibit hydrolytic activity with any of the cell-wall components. It binds at the interface between cellulose microfibrils and matrix polysaccharides in the cell wall, and is suggested to induce cell wall expansion by reversibly disrupting noncovalent bonds within this polymeric network (McQueen-Mason and Cosgrove, 1995, Plant Physiol. 107:87-100).
Some cellulose-binding organic substances alter cell growth and cellulose-microfibril assembly in vivo. Direct dyes, carboxymethyl cellulose (CMC) and fluorescent brightening agents (FBAs, e.g., calcofluor white ST) prevent Acetobacter xylinum microfibril crystallization, thereby enhancing polymerization. These molecules bind to the polysaccharide chains immediately after their extrusion from the cell surface, preventing normal assembly of microfibrils and cell walls (Haigler, 1991, "Relationship between polymerization and crystallization in microfibril biogenesis," in Biosynthesis and Biodegradation of Cellulose, pp. 99-124, Haigler and Weimer eds., Marcel Dekker, Inc., New York). Haigler discusses dyes and fluorescent brightening agents that bind to cellulose alter cellulose microfibril assembly in vivo. Modifications in cell shape were observed when red alga (Waaland and Waaland, 1975, Planta 126:127-138) and root tips (Hughes and McCully, 1975, Stain Technology 50:319-329) were grown in the presence of dyes. It is now evident that these molecules can bind to the cellulose chains immediately upon their extrusion from the cell surface of prokaryotes and eukaryotes (Haigler and Brown, 1979 Science 210:903-906; Benziman et al., 1980, Proc. Natl. Acad. Sci. USA 77:6678-6682; Haigler et al., 1980, Science 210:903-906; Brown et al., 1982, Science 218:1141-1142) and prevent crystal-structure formation (Haigler and Chanzy, 1988, J. Ultrastruct. Mol. Struct. Res. 98:299-311). In addition, the rate of cellulose polymerization was shown to increase in the presence of dye (Benziman et al., 1980). Crystallization was proposed to be the bottleneck in this coupled reaction and its prevention to result in accelerated cellulose synthase activity.
2.2. CELLULOSE BINDING PROTEINS AND DOMAINS
Many cellulases and hemicellulases (e.g., xylanases and mannases) have the ability to associate with their substrates. These enzymes typically have a catalytic domain containing the active site for substrate hydrolysis and a carbohydrate-binding domain or cellulose-binding domain (herein generally designated "CBD") for binding the insoluble cellulosic or hemicellulosic matrices.
To date, more than one hundred and twenty cellulose-binding domains (CBDs) have been classified into ten families designated I-X (Tomme et al., 1995, "Cellulose-Binding Domains: Classification and Properties", in ACS Symposium Series 618 Enzymatic Degradation and Insoluble Carbohydrates, pp. 142-161, Saddler and Penner eds., American Chemical Society, Washington, D.C.) (incorporated herein by reference). Most of the CBDs have been identified from cellulases and xylanases, but some are from other polysaccharides or from non-catalytic proteins. The CBDs identified thus far are from fungi, bacteria and slime molds.
The ten families of CBDs are as follows: family I CBDs are all from fungal .beta.-1,4-glycanases; family II CBDs are found in bacterial hydrolases; family III CBDs are found in .beta.-1,4-glucanases; family IV CBDs primarily have two conserved cysteine residues; family V is represented by a CBD from Erwinia chysanthemi; family VI CBDs are primarily from xylanases and nearly all located at the C-terminal end of the protein; family VII is represented by the CBD of Clostridium thermocellum; family VIII is represented by the CBD of Dictyostelium discoidum; family IX CBDs are all known to be present as tandem repeats at the C-terminal end of thermostable xylanases; and family X is represented by xylanase E from Pseudomonas florescens spp. cellulosa. For a detailed description of the CBD families and individual members useful in the present invention, see Table II of Tomme et al. which is incorporated herein by reference.
Shoseyov and Doi (1990, Proc. Natl. Acad. Sci. USA 87:2192-2195) isolated a unique cellulose-binding protein (CbpA) from the cellulase "complex" of the cellulolytic bacterium Clostridium cellulovorans. This major subunit of the cellulase complex was found to bind to cellulose, but had no hydrolytic activity, and was essential for the degradation of crystalline cellulose.
The cbpA gene has been cloned and sequenced (Shoseyov et al., 1992, Proc. Natl. Acad. Sci. USA 89:3483-3487). Using PCR primers flanking the cellulose-binding domain (herein, this specific CBD is designated "cbd") of CbpA, the latter was successfully cloned into an overexpression vector that enabled overproduction of the approximately 17 kDa cbd in Escherichia coli. The recombinant cbd exhibits very strong affinity to cellulose (U.S. Pat. No. 5,496,934; Goldstein et al., 1993, J. Bacteriol. 175:5762-5768; PCT International Publication WO 94/24158, all are incorporated by reference as if fully set forth herein).
In recent years, several CBDs have been isolated from different sources. Most of these have been isolated from proteins that have separate catalytic, i.e., cellulase and cellulose binding domains, and only two have been isolated from proteins that have no apparent hydrolytic activity but possess cellulose-binding activity (Goldstein et al., 1993, J. Bacteriol. 175:5762-5768; Morag et al., 1995, Appl. Environ. Microbiol. 61-1980-1986).
2.3. CLOSTRIDIUM CELLULOVORANS CBD EFFECTS ON SEEDLING AND POLLEN TUBE ELONGATION
The exogenous application of the cbd of Clostridium cellulovorans has been shown to modulate the elongation of pollen tubes and seedlings grown in culture. See PCT International Publication WO 94/24158 at pages 73-77.
The cbd of C. cellulovorans promoted pollen tube growth of peach pollen grains grown in liquid culture. Pollen grains exposed to 50 ug/ml of cbd produced pollen tubes almost twice size of pollen grains treated with bovine serum albumin (BSA) at 50 ug/ml.
Seeds of Arabidopsis thaliana germinated in distilled water in the presence of C. cellulovorans cbd responded differently to high versus low concentrations of cbd. High concentrations of cbd (1-100 ug/ml) dramatically reduced the root length. Low concentrations of cbd (1.times.10.sup.-6 to 1.times.10.sup.-4 ug/ml) promoted elongation of the roots whereas treatment with BSA had no effect. The effect on shoot length revealed a similar trend, but the differences between the treatments were not as dramatic as for the roots, and were not statistically different.
Cell walls of pollen tubes have been shown to contain exposed cellulose fibrils in the tip zone (reviewed by Steer and Steer, 1989, New Phytol. 111:323-358). Pollen tube elongation is known to be apical (reviewed by Cresti and Tiezzi, 1992, "Pollen tube emission, organization and tip growth", in Sexual Plant Reproduction, pp. 89-97, Cresti and Tiezzi eds., Springer-Verlag, Berlin). Gold-immunolabelling of cbd in pollen tubes revealed that cbd was present primarily at the tip zone. Moreover, the lack of calcofluor staining in the tip zone of cbd-treated pollen tubes indicated the absence of a crystalline structure. See PCT International Publication WO 94/24158.
It has already been established that XG chains cross-link the cellulosic network in the cell wall (reviewed by Roberts, 1994, Curr. Opin. Cell Biol. 6:688-694). It is accepted that a prerequisite for cell elongation is a loosening of the cross-linked cellulose network, by either hydrolysis as demonstrated by Inouhe and Nevins (1991, Plant Physiol. 96:426-431), transglycosylation (Fry et al., 1992, Biochem. J. 282:821-828; Nishitani and Tominaga, 1992, J. Biol. Chem. 267:21058-21064), or expansins that interact with the XG-cellulose bond (McQueen-Mason et al., 1992, The Plant Cell 4:1425-1433). By in vitro competition assays it was shown that cbd competes with XG for binding to cellulose. Maximum cbd binding to cellulose is achieved after 1 hour (Goldstein et al. 1993, J. Bacteriol. 175:5762-5768), compared to XG binding to cellulose that is achieved only after 4 hour (Hayashi et al. 1987, Plant Physiol. 83:384-389). It is suggested that, during the elongation process, cellulose microfibrils become exposed and cbd competes with XG on binding to the exposed cellulose microfibril. It is therefore possible that this competition results in a temporary loosening of the cell wall and consequently enhanced elongation.
The inhibitory effect of cbd on root elongation can be explained by steric hindrance of the cellulose fibrils by excess amounts of cbd, which block access for enzymes or other proteins that modulate cell elongation via loosening, of the rigid cellulose-fibril network. This hypothesis is supported by Nevins, who prevented auxin-induced elongation with anti-.beta.-D-glucan antibodies (Hoson and Nevins, 1989, Plant Physiol. 90:1353-1358) or with antibodies specific to cell wall glucanases (Inouhe and Nevins, 1991, Plant Physiol. 96:426-431).
The cbd of the CbpA protein of C. cellulovorans is a bacterial protein. Its mode of action in modulating cell wall elongation may be different from that of the natural process.